CN113667659A - Polypeptide with polyester degradation activity and application thereof - Google Patents

Abstract

The present invention relates to polypeptides having polyester degrading activity and uses thereof. In particular, the present invention relates to a novel isolated polypeptide comprising an amino acid sequence having at least 94%, 95%, 99% or 100% identity to the full length amino acid sequence as set forth in SEQ ID No. 1 and having polyester degrading activity and uses thereof.

Description

Polypeptide with polyester degradation activity and application thereof

The application is a divisional application of the application of international application date 2015, 10-20 days, international application number PCT/EP2015/074222, 4-20 days in 2017, entering the Chinese national stage, application number 201580057013.5, invention name 'polypeptide with polyester degradation activity and application thereof'.

Technical Field

The present invention relates to a novel polypeptide having enzymatic activity and use thereof. The invention also relates to methods of producing the polypeptides, encoding nucleic acid molecules, recombinant cells, and methods of using the polypeptides to degrade polyester-containing materials. The polypeptides of the invention are particularly suitable for degrading polylactic acid and polylactic acid containing materials like plastic materials.

Background

Polyesters are used in particular in the form of plastic materials in a large number of technical fields ranging from food packaging to the medical field, clothing, the automotive industry, etc. As an example, certain polyesters (e.g., polyethylene terephthalate-PET, polylactic acid-PLA, etc.) are used in the manufacture of garments and packaging, but also in the form of thermosetting resins for use in the manufacture of automobiles or other parts.

Thus, over the last decades, the production of polyester-containing plastics has increased significantly. More than 50% of these plastics are used in single use disposable applications such as packaging, agricultural films, disposable consumer products or for short term products that are discarded within 1 year after manufacture. Unfortunately, plastics can persist for decades depending on local environmental factors such as uv light exposure level, temperature, presence of suitable microorganisms, and the like. Thus, worldwide, a large amount of plastics is accumulated in landfills and natural habitats, creating increasing environmental problems.

One solution to reduce the environmental and economic impact associated with the accumulation of plastic is recycling, where the plastic material is mechanically reprocessed to make new products. However, the actual recycling process uses a large amount of electricity, particularly during the extrusion step, and the equipment used is also expensive, resulting in a high price that may not be competitive compared to virgin plastic.

Another potential method for recycling plastics consists of chemical recycling which allows the chemical components of the polymer to be recovered. The resulting monomers can then be used to remanufacture plastics or to prepare other synthetic chemicals. However, the recycling process has so far only been performed on purified polymers and is not efficient on original plastic articles consisting of a mixture of crystalline and amorphous polymers and additives. Furthermore, the recycling process is expensive, resulting in non-competitive monomers compared to the original monomers.

On the other hand, enzymatic degradation is considered an ideal waste treatment method, as enzymes can accelerate hydrolysis of plastics and can be incorporated into the natural circulation of organic materials. In addition, the hydrolyzates (i.e., monomers and oligomers) can be recycled as material for the polymers. Thus, depolymerization of polymers contained in plastic articles by enzymes is of great interest as an alternative to existing and unsatisfactory processes.

However, this approach has not heretofore led to the implementation of an efficient and industrial enzymatic process for degrading polyester-containing materials.

Indeed, many bacteria are known to be capable of degrading polyesters. For example, as for polylactic acid, there are reports of degradative enzymes derived from Actinomycetes (Actinomycetes) such as Amycolatopsis sp (strain K104-1) and from Paenibacillus amyloliquefaciens (strain TB-13). However, to date, the identified polypeptides have poor degradability and only allow for degradation of the polymer in emulsion form. There are a limited number of reports of microorganisms capable of degrading polyester-containing materials in the form of films or pellets, and furthermore, their enzymes are not well understood.

In view of the foregoing, there is a need for novel enzymes that are active in degrading polyesters, and more particularly polyesters contained in plastic articles.

Disclosure of Invention

The studies carried out by the applicant have led to the identification of a novel polypeptide derived from Actinomadura sp and having polyester degrading activity. This polypeptide has never been reported or isolated in the art and represents a substantial improvement in the development of an industrial process for degrading polyester-containing materials.

The invention is based in particular on the identification of this novel polypeptide having excellent properties for degrading polyesters. The present invention relates to a solution to obtain, on an industrial scale, the degradation of polyesters contained in plastic articles, the degradation products (monomers and oligomers) of which can be reused for economically and reliably producing new polyesters.

Thus, the present invention relates to novel polypeptides having enzymatic activity, their manufacture and use. The invention also relates to nucleic acids encoding these polypeptides, vectors, recombinant cells expressing these polypeptides and uses thereof. The invention further relates to compositions comprising at least one polypeptide of the invention and methods for producing target oligomers and/or monomers from polyester-containing materials, such as plastic articles made from polyesters. The invention also relates to biodegradable plastic compounds or plastic articles containing at least one of these polypeptides and/or recombinant cells expressing these polypeptides.

Accordingly, an object of the present invention relates to an isolated polypeptide comprising an amino acid sequence having at least 94%, 95%, 99% or 100%, preferably at least 95%, 96%, 97%, 98%, 99% or 100% identity to the full length amino acid sequence as set forth in SEQ ID No. 1 and having polyester degrading activity.

In a particular embodiment, the amino acid residue sequence of the polypeptide differs from SEQ ID No. 1 due to amino acid residue substitutions of amino acid residues at one or more positions. In a preferred embodiment, the amino acid residue substitution introduces a cysteine or additional salt bridge in the amino acid residue sequence and thereby increases the thermostability of the polypeptide compared to the thermostability of the native polypeptide (i.e., the polypeptide having the amino acid residue sequence shown in SEQ ID NO: 1).

Another object of the present invention is to provide a polypeptide comprising an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 5.

In a particular embodiment, the polypeptide comprises one or several glycosylated amino acid residues.

Another object of the invention is a nucleic acid encoding a polypeptide as defined above. The invention also relates to an expression cassette comprising a nucleic acid as defined above and to a vector comprising a nucleic acid or an expression cassette as defined above.

The invention also relates to a recombinant cell or host cell, preferably a recombinant microorganism, containing at least one nucleic acid or expression cassette or vector as defined above, and to extracts thereof preferably exhibiting enzymatic activity.

It is another object of the invention to provide a method for producing a polypeptide of the invention comprising (i) culturing a recombinant cell as defined above, (ii) recovering the culture supernatant, and optionally (iii) isolating or purifying the polypeptide.

The invention also discloses a composition comprising a polypeptide as defined above or a recombinant cell expressing said polypeptide or an extract thereof.

The invention further relates to the use of a polypeptide, corresponding nucleic acid, expression cassette, vector, recombinant cell extract or composition as defined above for the enzymatic degradation of a polyester containing material, preferably a PLA containing material, even more preferably a PLLA containing material.

It is a further object of the present invention to provide a method for degrading a polyester containing material, wherein the polyester containing material is contacted with a polypeptide, a corresponding nucleic acid, an expression cassette, a vector, a recombinant cell or a recombinant cell extract or composition as defined above. The method advantageously further comprises the step of collecting the resulting monomers and/or oligomers.

The invention also relates to a method for producing monomers and/or oligomers from a polyester containing material, comprising exposing the polyester containing material to a polypeptide, a corresponding nucleic acid, an expression cassette, a vector, a recombinant cell or a recombinant cell extract or composition as defined above, and optionally recovering the monomers and/or oligomers.

It is a further object of the present invention to provide a polyester-containing material comprising a polypeptide as defined above and/or recombinant cells expressing said polypeptide.

The invention also provides a method for producing said polyester-containing material, comprising the step of mixing a polyester and a polypeptide as defined above and/or recombinant cells expressing said polypeptide, wherein said mixing step is performed at a temperature at which said polyester is in a partially or fully molten state, preferably during an extrusion process.

The invention further relates to the use of a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% identity to the full length amino acid sequence as set forth in SEQ ID No. 1 and having polyester degrading activity for degrading polyester containing material.

Drawings

FIG. 1 is a photograph of an SDS-Page gel of pH 10 flow-through obtained by anionic purification, showing that the molecular weight of the polypeptide of the invention is 27 kDa;

FIG. 2. amino acid sequence of the polypeptide of the invention (SEQ ID NO:1), in which the most important residues are highlighted;

FIG. 3: graphs showing polyesterase catalyzed hydrolysis of Natureplast PLLA powder (33g/L) and lactic acid production as a function of PLLA particle size;

FIG. 4: indicating polyesterase catalysis

7001D PLA powder (33 g-L) hydrolysis and lactic acid production as a function of PLA particle size;

FIG. 5: shows a graph of polyesterase catalyzed hydrolysis of PLA film (17g/L) and lactic acid production;

FIG. 6: a graph showing the hydrolysis and lactic acid production of polyesterase catalyzed PLA commercial articles (PLA cups, plates, films and tableware) (33 g/L).

FIG. 7: showing PLA in the presence of CaCO₃And Ca (OH)₂The medium of (1) is hydrolyzed.

FIG. 8: a graph showing the hydrolysis of PLA-containing material containing 96% PLA and 4% of the polypeptide of the invention in Tris buffer at pH 9.5 at 28 ℃, 37 ℃ and 45 ℃ and a control hydrolysis containing 100% PLA.

Detailed Description

The present invention relates generally to an isolated polypeptide comprising at least a biologically active portion of the amino acid sequence shown as SEQ ID No. 1, which is capable of depolymerizing a polyester, more preferably a polylactic acid. Preferably, this polypeptide, which is active in the temperature range of 20 ℃ to 90 ℃ and at least 20 ℃ to 60 ℃, is useful for degrading polyester plastic materials. This polypeptide or its encoding nucleic acid sequence may also be used to generate recombinant microorganisms that may be used to cause degradation of polyester-containing materials. The recombinant microorganism may further exhibit natural or recombinant polymer synthesis activity such that the microorganism is able to reuse monomers and/or oligomers resulting from polyester degradation.

The following is a description of the invention, including its preferred embodiments, given in a generic manner. The invention is further exemplified by the disclosure given below under the heading "examples" which provides experimental data supporting the invention and means to carry out the invention.

Definition of

The present disclosure will be best understood by reference to the following definitions.

The term "isolated" or "isolating" means that a substance is removed from its original environment (e.g., the natural environment). For example, an isolated polypeptide typically lacks at least some of the polypeptide or other components of the cell with which it is normally associated or with which it is normally admixed or solubilized. Isolated polypeptides include the naturally occurring polypeptides, recombinant polypeptides, polypeptides expressed or secreted by a host cell, and polypeptides in a host cell or culture or extract thereof, in purified or partially purified form. In a preferred aspect, the polypeptide is at least 10% pure, preferably at least 50% pure, more preferably at least 60%, 70%, 80%, 90% pure, as determined by SDS-PAGE. A purity of less than 100% means herein that the polypeptide preparation contains other polypeptide material with which it is naturally or recombinantly associated. With respect to a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its native genomic context (e.g., in a vector, an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

The term "modification" means herein any chemical modification of a polypeptide consisting of SEQ ID NO:1 or homologous sequences thereof and the genetic manipulation of DNA encoding such a polypeptide. The modification may be a substitution, deletion and/or insertion of one or several amino acids. Thus, the terms "mutant" and "variant" are used interchangeably to refer to a polypeptide consisting of SEQ ID NO:1 having identified amino acid substitutions, deletions and/or insertions at defined residues.

The term "glycosylation" means that the substance comprises one or several glycans attached to amino acid residues of the polypeptide. In the context of the present invention, glycosylation encompasses N-linked glycans attached to the amide nitrogen of an asparagine residue, O-linked glycans attached to the hydroxyl oxygen of a serine or tyrosine residue, C-linked glycans attached to the carbon of a tryptophan residue.

The term "recombinant" refers to a nucleic acid construct, vector, polypeptide, or cell produced by genetic engineering.

As used herein, the term "sequence identity" or "identity" refers to the number (%) of matches (identical amino acid residues) in each position obtained from aligning two polypeptide sequences. Sequence identity is determined by comparing sequences when aligned so as to maximize overlap and identity while minimizing the number of sequence gaps. In particular, depending on the length of the two sequences, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms. Sequences of similar length are preferably aligned using an overall alignment algorithm (e.g., Needleman and Wunsch algorithms; Needleman and Wunsch,1970) that optimally aligns the sequences over the entire length, while sequences of substantially different length are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithms (Smith and Waterman,1981) or Altschul algorithms (Altschul et al, 1997; Altschul et al, 2005)). Alignment for the purpose of determining percent amino acid sequence identity may be accomplished in a variety of ways that are well within the skill of the art, for example, using publicly available computer software available on Internet websites such as http:// blast. ncbi. nlm. nih. gov/or http:// www.ebi.ac.uk/Tools/emboss /). One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the compared sequences. For purposes herein, amino acid sequence identity% values refer to values generated using the paired sequence alignment program EMBOSS Needle that generates an optimal overall alignment of two sequences using the Needleman-Wunsch algorithm, with all search parameters set to default values, i.e., scoring matrix BLOSUM62, gap open 10, gap extension 0.5, end gap penalty, end gap open 10, and end gap extension 0.5.

The term "expression" as used herein refers to any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Herein, the terms "peptide", "polypeptide", "protein" and "enzyme" are used interchangeably and refer to a chain of amino acids linked by peptide bonds, irrespective of the number of amino acids forming said chain. Amino acids are herein represented by their single or three letter codes according to the following nomenclature: a: alanine (Ala); c: cysteine (Cys); d: aspartic acid (Asp); e: glutamic acid (Glu); f: phenylalanine (Phe); g: glycine (Gly); h: histidine (His); i: isoleucine (Ile); k: lysine (Lys); l: leucine (Leu); m: methionine (Met); n: asparagine (Asn); p: proline (Pro); q: glutamine (Gln); r: arginine (Arg); s: serine (Ser); t: threonine (Thr); v: valine (Val); w: tryptophan (Trp) and Y: tyrosine (Tyr).

In the context of the present invention, "polyester-containing material" refers to an article, such as a plastic article, comprising at least one polyester in crystalline, semi-crystalline or completely amorphous form. In a particular embodiment, polyester-containing material refers to any article made of at least one plastic material, such as plastic sheets, tubes, rods, profiles, molds, films, blocks, etc., containing at least one polyester and possibly other substances or additives such as plasticizers, inorganic or organic fillers. In a particular embodiment, the polyester-containing material comprises a polyester and at least one additional polymer, such as a polyolefin, disposed relative to each other in such a way that they cannot be easily separated. Preferably, the polyester-containing material consists of a mixture of crystalline and amorphous polyesters, and/or semi-crystalline polyesters and additives. More preferably, the polyester-containing material is a plastic article of manufacture, such as packaging, agricultural films, disposable items, and the like. In another particular embodiment, polyester-containing material refers to a plastic compound or plastic formulation suitable for making plastic articles in a molten or solid state. In the context of the present invention, a plastic compound encompasses a homogeneous blend of at least one polyester and at least one polypeptide of the invention and/or recombinant cells expressing said polypeptide, wherein said polypeptide and/or recombinant cells are capable of degrading said polyester. Preferably, the plastic compound consists of a semi-crystalline and/or amorphous polymer, or a mixture of semi-crystalline polymers and additives.

In the present description, "polyester" encompasses polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene terephthalate-co-isosorbide terephthalate (PEIT), polylactic acid (PLA), poly (L-lactic acid) (PLLA), poly (D-lactic acid) (PDLA), poly (D, L-lactic acid) (PDLLA), PLA stereocomplex (scPLA), Polyhydroxyalkanoate (PHA), poly (3-hydroxybutyrate) (P (3HB)/PHB), poly (3-hydroxyvalerate) (P (3HV)/PHV), poly (3-hydroxyhexanoate) (P (3HHx)), poly (3-hydroxyoctanoate) (P (3HO)), poly (3-hydroxydecanoate) (P (HD 3)), poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (P (3 HB-co-3-hydroxyvalerate) (P (3HHx)) -3HV)/PHBV), poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (P (3 HB-co-3 HHx)/(phbhxx)), poly (3-hydroxybutyrate-co-5-hydroxyvalerate) (PHB5HV), poly (3-hydroxybutyrate-co-3-hydroxypropionate) (PHB3HP), polyhydroxybutyrate-co-hydroxyoctanoate (PHBO), polyhydroxybutyrate-co-hydroxyoctadecanoate (PHBOd), poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) (P (3 HB-co-3 HV-co-4 HB)), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), Polybutylene adipate-co-terephthalate (PBAT), polyethylene furanoate (PEF), Polycaprolactone (PCL), poly (ethylene adipate) (PEA), and blends/mixtures of these polymers.

"Polymer" refers to a compound or mixture of compounds whose structure is made up of a plurality of repeating units joined by covalent chemical bonds. In the context of the present invention, the term polymer includes natural or synthetic polymers composed of a single type of repeating unit (i.e. homopolymers) or of a mixture of different repeating units (i.e. copolymers).

According to the invention, "oligomer" means a molecule containing from 2 to about 20 monomer units.

Novel isolated polypeptides

The present invention relates to a novel polypeptide capable of degrading plastics having an ester bond in their molecular structure. More particularly, the present invention discloses a newly identified and isolated polypeptide exhibiting polyesterase activity. The polypeptide was originally isolated from a native bacterial strain of keratin-degrading Actinomadura keratinilytica T16-1 or DSMZ 45195. It is contemplated that the polypeptides of the invention are capable of hydrolyzing ester bonds in natural and artificial polyesters.

In a first aspect, the present invention relates to an isolated polypeptide comprising an amino acid sequence having at least 94%, 95%, 99% or 100% identity to the full length amino acid sequence as set forth in SEQ ID No. 1 provided below, and having polyester degrading activity.

Advantageously, the polypeptide comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the full-length amino acid sequence as set forth in SEQ ID NO 1.

SEQ ID NO:1：

ATQNNPPSWGLDRIDQTNLPLSRSYTYNSTGAGVNAYIIDTGIYTAHSDFGGRATNVYDALGGNGQDCNGHGTHVAGTVGGAAYGVAKAVNLRGVRVLNCSGSGTTSGVIAGMNWVASNHVKPAVANMSLGGGYSSSLNTAANNLASSGVFLAVAAGNETTNACNRSPASAANATTVAASTSTDARASYSNYGSCVHLYAPGSSITSAWLNGGTNTISGTSMATPHVAGTAALYKATYGDASFSTIRSWLVSNATSGVITGNVSGTPNLLLNKRSL

As used herein, a polypeptide of the present invention may also be referred to as a polypeptide having polyesterase activity, or interchangeably as a polyesterase.

It is a particular object of the invention to provide an isolated polypeptide comprising an amino acid sequence having at least 94%, 95%, 99% or 100% identity to the full length amino acid sequence as set forth in SEQ ID No. 1 and having polylactic acid degrading activity, and more preferably poly- (L-lactic acid) degrading activity. In a particular embodiment, the polypeptide comprises an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full-length amino acid sequence set forth in SEQ ID No. 1.

In a particular embodiment, the isolated polypeptide comprises all or a biologically active portion of the amino acid sequence set forth in SEQ ID NO. 1. More specifically, a "biologically active portion" of a polypeptide designates that portion of that polypeptide which confers or exhibits polyesterase activity for the entire polypeptide. The active moiety may, for example, confer specificity or affinity to a substrate, and it may contain a catalytic site. An active portion of a polypeptide also designates the mature form of the polypeptide (i.e., it does not contain a signal peptide at the N-terminus of the polypeptide). In one embodiment, the biologically active portion comprises at least a portion of the amino acid sequence set forth in SEQ ID No. 1, said at least a portion comprising the amino acids His 71, Asp 40 and Ser 221 forming the catalytic site of the polypeptide. Alternatively, or in addition, the active moiety advantageously comprises the amino acids Ala 172, Ala 174 and His 197 and/or the amino acids Asp 12, Asp 15 and Gln 16 forming a calcium binding site and/or the amino acids Cys 68-Cys 100 and Cys 164-Cys 195 forming a disulfide bond and/or the amino acids forming a polyester binding site. In a particular embodiment, the biologically active portion comprises or consists of amino acids 12 to 221 of SEQ ID No. 1. In another embodiment, the biologically active portion comprises or consists of amino acids 40 to 221 of SEQ ID No. 1.

Another object of the present invention is to provide a polypeptide comprising or consisting of the amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 5. Another object of the present invention is to provide a peptide comprising or consisting of amino acids 1 to 29 of the peptide signal of SEQ ID NO. 5 corresponding to a polypeptide.

SEQ ID NO:5：

MRRRTLPIAVLAAVPLAVAGALPAGAAPAAPAVPVAMAAAGQGVAGQYIVTLKKGVSVDSTVAKRGIRTQHRFGKVLNGFSAKLTDDQLSKLRTTPGVASIEQDAVITVDATQNNPPSWGLDRIDQTNLPLSRSYTYNSTGAGVNAYIIDTGIYTAHSDFGGRATNVYDALGGNGQDCNGHGTHVAGTVGGAAYGVAKAVNLRGVRVLNCSGSGTTSGVIAGMNWVASNHVKPAVANMSLGGGYSSSLNTAANNLASSGVFLAVAAGNETTNACNRSPASAANATTVAASTSTDARASYSNYGSCVHLYAPGSSITSAWLNGGTNTISGTSMATPHVAGTAALYKATYGDASFSTIRSWLVSNATSGVITGNVSGTPNLLLNKRSL

Another object of the present invention is to provide variants of the polypeptide as shown in SEQ ID NO. 1 comprising substitutions, deletions and/or insertions of one or several amino acid residues of the polypeptide of SEQ ID NO. 1, having polyester degrading activity.

In a particular embodiment, the variant exhibits greater thermostability as compared to the native polypeptide. For example, disulfide bonds are introduced by substitution and/or insertion of amino acid residues, thereby creating additional cysteine residues in the amino acid sequence compared to the native amino acid sequence. Alternatively or additionally, additional salt bridges may be introduced in the amino acid sequence of the polypeptide.

In a particular embodiment, the variant comprises one or several substitutions selected from T175C, R247C, N139D, S170R, N143R, N173E, S194P, H197D, L210P, G212N, I217K, R166K, T160A, L138A or combinations thereof, relative to SEQ ID No. 1, and has polyester degrading activity, preferably PLA degrading activity.

In a particular embodiment, the variant comprises or consists of the amino acid sequence SEQ ID NO:1 with substitutions T175C and R247C and has polyester degrading activity, preferably PLA degrading activity.

In another particular embodiment, the variant comprises or consists of the amino acid sequence SEQ ID NO 1 with the substitutions N139D and S170R and has polyester degrading activity, preferably PLA degrading activity.

In another particular embodiment, the variant comprises or consists of the amino acid sequence SEQ ID NO 1 with substitutions N143R and N173E and has polyester degrading activity, preferably PLA degrading activity.

In a particular embodiment, the variant comprises or consists of the amino acid sequence SEQ ID No. 1 with the substitutions T175C, R247C, N139D, S170R, N143R, N173E, S194P, H197D, L210P, G212N, I217K, R166K, T160A and L138A and has polyester degrading activity, preferably PLA degrading activity.

In a particular embodiment, the variant comprises up to 14 amino acid residue substitutions compared to the amino acid sequence as shown in SEQ ID No. 1 and has polyester degrading activity, preferably PLA degrading activity. In another embodiment, the variant comprises up to 17 amino acid residue substitutions compared to the amino acid sequence as set forth in SEQ ID No. 1.

Advantageously, the variant has better polyester degrading activity than the native polypeptide of SEQ ID NO: 1. More preferably, the variant polypeptide has greater stability at high temperatures than the native polypeptide of SEQ ID NO: 1.

In another embodiment, the polypeptide comprises one or several glycosylations. For example, the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO 1, wherein at least one amino acid residue is glycosylated. Advantageously, the glycosylated polypeptide exhibits greater stability, in particular greater thermostability, compared to the native polypeptide (the unglycosylated polypeptide of SEQ ID NO: 1). For example, at least one asparagine residue of the amino acid sequence SEQ ID No. 1 is glycosylated and an oligosaccharide is attached at the amide nitrogen of said asparagine residue. More particularly, SEQ ID No. 1 comprises at least one glycosylation of an amino acid residue selected from: n28, N99, N127, N158, N165, N173, N253, N262, or a combination thereof. In a preferred embodiment, SEQ ID NO 1 comprises at least one glycosylation of an amino acid residue selected from the group consisting of: n28, N158, N165.

The polypeptides of the invention are particularly active at temperatures ranging from 20 ℃ to 90 ℃, preferably from 20 ℃ to 60 ℃, more preferably from 30 ℃ to 55 ℃, even more preferably from 40 ℃ to 50 ℃, even more preferably at 45 ℃. In a particular embodiment, the polypeptide is still active at a temperature between 60 ℃ and 90 ℃, preferably at 80 ℃.

Similarly, the polypeptides of the invention are particularly active in the pH range of 5 to 11, preferably in the pH range of 7 to 10, more preferably in the pH range of 8.5 to 9.5, even more preferably in the pH range of 8 to 9.

The isolated polypeptide of the invention advantageously has at least 0.02g.mg^-1.h^-1、0.05g.mg^-1.h^-1、0.1g.mg^{- 1}.h^-1、0.15g.mg^-1.h^-1、0.2g.mg^-1.h^-1、0.5g.mg^-1.h^-1、1g.mg^-1.h^-1、1.5g.mg^-1.h^-1Or 2g.mg^-1.h^-1The productivity of (c). By "productivity" it is meant the amount of degradation products (i.e. monomers) formed per unit of polypeptide and per unit of time at a pH comprised between 8 and 9 and at a temperature of 45 ℃ +/-5 ℃.

The polypeptides of the invention are particularly useful for degrading polylactic acid (PLA), and more particularly poly (L-lactic acid) (PLLA).

In a particular embodiment, the polypeptides of the invention are enantiospecific. This means that the polypeptide is capable of acting on the L-enantiomer in the polyester, but not on the D-enantiomer, or vice versa.

The polypeptide of the invention may be produced by recombinant techniques, or it may be isolated or purified from a natural source (i.e., a microorganism and more particularly a bacterium, yeast or fungus), or it may be produced artificially. In the context of the present invention, the term "derived from a microorganism" in relation to a polypeptide indicates that the polypeptide has been isolated from such a microorganism or that the polypeptide comprises all or a biologically active part of the amino acid sequence of the polypeptide isolated or characterized from such a microorganism. More particularly, the polypeptide of the invention may be produced by recombinant Bacillus (Bacillus), recombinant escherichia coli (e.coli) or recombinant Yarrowia lipolytica (Yarrowia lipolytica).

The polypeptides of the invention can be purified by techniques known per se in the art, such as chromatography (e.g., ion exchange, affinity, size exclusion, reverse phase, etc.) and precipitation (e.g., salting out, isoelectric point, organic solvents, non-ionic hydrophilic polymers, etc.), and stored by conventional techniques. The polypeptide may be further modified to improve, for example, its stability or activity. It can be used as such, in purified form, alone or in combination with additional enzymes, for catalyzing enzymatic reactions involved in the degradation and/or recycling of polyester-containing materials. The polypeptide may be in dissolved form or on a solid phase. In particular, it may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastics, polymers, filters, membranes, e.g. in the form of beads, columns, plates, etc.

It is another object of the present invention to provide a composition comprising an isolated polypeptide of the present invention and/or a corresponding nucleic acid, expression cassette, vector, recombinant cell or recombinant cell extract, and optionally, additives, excipients, and the like. In the context of the present invention, the term "composition" encompasses all kinds of compositions comprising the polypeptide of the present invention in isolated or at least partially purified form. The composition may be liquid or dry, for example in powder form. In some embodiments, the composition is a lyophilizate. For example, a composition may comprise a polypeptide of the invention and/or a recombinant cell encoding the polypeptide or an extract thereof, and optionally, excipients and/or agents, and the like. Suitable excipients encompass buffers commonly used in biochemistry; an agent for adjusting pH; preservatives (preservative) such as sodium benzoate, sodium sorbate or sodium ascorbate; a preservative (preservative); protective or stabilizing agents such as starch, dextrin, gum arabic, salt, sugar such as sorbitol, trehalose or lactose, glycerol, polyethylene glycol, polypropylene glycol, propylene glycol; chelating agents such as EDTA; an amino acid; a carrier such as a solvent or aqueous solution; and the like. The composition of the invention may be obtained by mixing the polypeptide with one or several excipients.

The composition of the invention may comprise from 0.1% to 90%, preferably from 0.1% to 50%, more preferably from 0.1% to 30%, even more preferably from 0.1% to 5% by weight of the polypeptide of the invention, and from 10% to 99.9%, preferably from 50% to 99.9%, more preferably from 30% to 99.9%, even more preferably from 95% to 99.9% by weight of the excipient. A preferred composition comprises between 0.1 and 5% by weight of the polypeptide of the invention.

In a particular embodiment, the composition may further comprise an additional polypeptide exhibiting enzymatic activity. The amount of the polypeptide of the invention will be readily adapted by the skilled person depending on, for example, the nature of the polyester containing material to be degraded and/or the additional enzyme/polypeptide contained in the composition.

In a particular embodiment, the isolated polypeptide of the invention is dissolved in an aqueous medium together with one or several excipients, in particular excipients capable of stabilizing or protecting the polypeptide against degradation. For example, the polypeptides of the invention may be dissolved in water and eventually combined with additional components such as glycerol, sorbitol, dextrin, starch, glycols such as propylene glycol, salts, and the like. The resulting mixture may then be dried so as to obtain a powder. Methods for drying the mixture are well known to those skilled in the art and include, without limitation, lyophilization, freeze drying, spray drying, supercritical drying, downdraft evaporation, thin layer evaporation, centrifugal evaporation, conveyor belt drying, fluid bed drying, drum drying, or any combination thereof.

In another specific embodiment, the composition of the invention comprises at least one recombinant cell expressing a polypeptide of the invention or an extract thereof. "extract of a cell" designates any fraction obtained from a cell that is substantially free of living cells, such as cell supernatant, cell debris, cell walls, DNA extract, enzymes or enzyme preparations or any preparation obtained from a cell by chemical, physical and/or enzymatic treatment. Preferably the extract is an enzymatically active extract. The composition of the invention may comprise one or several recombinant cells of the invention or extracts thereof, and optionally one or several additional cells.

In a particular embodiment, the composition consists of or comprises a lyophilization medium for a recombinant microorganism expressing and secreting a polypeptide of the invention. In a particular embodiment, the powder comprises a polypeptide of the invention and a stabilizing/solubilizing amount of glycerol, sorbitol or dextrin such as maltodextrin and/or cyclodextrin, starch, glycol such as propylene glycol, and/or salt.

In another embodiment, the polypeptide of the invention is immobilized on a solid support. The polypeptide may be immobilized by any suitable method described in the prior art, such as covalent binding, adsorption, entrapment or membrane confinement. A wide variety of vectors may be used to immobilize the polypeptides of the invention. The vector to be selected depends on the intended use. Suitable supports include, without limitation, plastics, metals, inorganic supports such as glass, silica, alumina, bentonite, hydroxyapatite, nickel/nickel oxide, titanium, zirconia, polymeric supports, and the like. The carrier may be in the form of a surface, a powder, micro-or nanobeads, a gel, a solvent-swollen or water-swollen gel or matrix, a mesh matrix or gel, a membrane, a fibrous carrier, a porous carrier, or the like. Methods for immobilizing polypeptides are well known to the skilled artisan (see, e.g., Tischer and Wedekind, Topics in Current Chemistry,1999,200,95-126, and Alloue et al, Biotechnol Agron Soc Environ 2008,12, 57-68; the disclosure of which is incorporated herein by reference).

Once prepared, the support of the invention can be used directly in the reaction medium. In other words, the support of the invention may be added only in the reaction medium. When the carrier is solvent swellable, the solvent of the reaction may be selected so as to provide adequate carrier swelling to render accessible the immobilized polypeptide without compromising the catalytic activity of the polypeptide. As an alternative, the support may be used in a preparation reactor, which may be, for example, an enzyme reactor, a membrane reactor, a continuous flow reactor such as a stirred tank reactor, a continuously operated packed bed reactor, or a continuously operated fluidized bed reactor, or a packed bed reactor. In some embodiments, the carriers of the present invention are recyclable and can be used several times in succession.

Nucleic acids

Another object of the invention is a nucleic acid encoding a polypeptide as defined above.

As used herein, the terms "nucleic acid," "nucleic acid sequence," "polynucleotide," "oligonucleotide," and "nucleotide sequence" are used interchangeably and refer to a sequence of deoxyribonucleotides and/or ribonucleotides. The nucleic acid may be DNA (cDNA or gDNA), RNA, or a mixture of both. It may be in single stranded form or in duplex form, or a mixture of both. It may be of recombinant, artificial and/or synthetic origin and it may comprise modified nucleotides comprising, for example, a modified bond, a modified purine or pyrimidine base, or a modified sugar.

The nucleic acids of the invention may be in isolated or purified form and prepared, isolated and/or manipulated by techniques known per se in the art, such as cloning and expressing a cDNA library, amplification, enzymatic synthesis or recombinant techniques. Nucleic Acids can also be synthesized in vitro by well-known chemical synthesis techniques as described, for example, in Belouov (1997) Nucleic Acids Res.25: 3440-3444.

The invention also encompasses nucleic acids that hybridize under stringent conditions to nucleic acids encoding a polypeptide as defined above. Preferably, the stringent conditions include incubating the hybridization filter in 2 XSSC/0.1% SDS at about 42 ℃ for about 2.5 hours, followed by washing the filter 4 times in 1 XSSC/0.1% SDS at 65 ℃ for 15 minutes each. Protocols used are described in bibliography such as Sambrook et al (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).

The invention also encompasses nucleic acids encoding the polypeptides of the invention, wherein the sequence of said nucleic acid or at least a portion of said sequence has been engineered using optimized codon usage.

A particular embodiment of the invention consists in an isolated nucleic acid encoding a polypeptide as defined above, comprising the sequence shown in SEQ ID NO 2 as disclosed below.

SEQ ID NO:2：

5’gccacgcagaacaacccgccgtcgtggggcctggaccgcatcgaccagacgaacctgccgctgtcgcgcagctacacctacaattccaccggcgcgggcgtgaacgcctacatcatcgacaccggcatctacaccgcgcactccgacttcggcggccgcgccaccaacgtctacgacgccctcggcggcaacggccaggactgcaacggccacggcacccacgtcgcgggcaccgtcggcggcgccgcctacggcgtggccaaggcggtcaacctgcgcggcgtgcgcgtgctcaactgcagcggcagcggcaccacctccggtgtcatcgccggcatgaactgggtggccagcaaccacgtcaagcccgccgtggcgaacatgtcgctgggcggcggctactcctcctccctgaacacggccgccaacaacctggccagctccggcgtgttcctggccgtcgccgcgggcaacgagaccaccaacgcctgcaaccgctcgccggccagcgccgccaacgccaccacggtcgccgcgagcaccagcaccgacgcccgggcctcctacagcaactacggctcgtgcgtccacctgtacgcgcccggctcgtccatcacctccgcctggctgaacggcggcaccaacaccatcagcggcacgtcgatggccacgccgcacgtggccgggaccgccgccctctacaaggcgacctacggcgacgcctcgttcagcaccatccgcagctggctggtcagcaacgccacctccggcgtcatcaccggcaacgtgtcgggcaccccgaacctgctgctgaacaagcgctccctg 3’

Alternatively, the nucleic acid of the invention may be deduced from the sequence of the polypeptide of the invention and the codon usage may be adapted depending on the host cell in which the nucleic acid is to be transcribed. These steps can be performed according to methods well known to the person skilled in the art, and some of said methods are described in the reference manual Sambrook et al (Sambrook et al, 2001).

The nucleic acids of the invention may further comprise additional nucleotide sequences useful for causing or regulating expression of the polypeptide in a selected host cell or system, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides, and the like.

The present invention further relates to an expression cassette comprising a nucleic acid of the invention operably linked to one or more control sequences which direct the expression of the nucleic acid in a suitable host cell. Typically, an expression cassette comprises or consists of a nucleic acid of the invention operably linked to a transcription promoter and a transcription terminator.

The invention also relates to a vector comprising a nucleic acid or expression cassette as defined above.

The term "vector" refers to a DNA molecule that serves as a vehicle for transferring recombinant genetic material into a host cell. The main types of vectors are plasmids, phages, viruses, cosmids and artificial chromosomes. The vector itself is typically a DNA sequence consisting of an insert (heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of transferring genetic information to a vector in a host is generally to isolate, propagate, or express the insert in the target cell. Vectors referred to as expression vectors (expression constructs) are particularly suitable for expressing heterologous sequences in target cells, and typically have a promoter sequence that drives expression of the heterologous sequence encoding the polypeptide. Typically, the regulatory elements present in an expression vector include a transcription promoter, a ribosome binding site, a terminator and, optionally, an operator. Preferably, the expression vector also contains an origin of replication for autonomous replication in the host cell, a selectable marker, a limited number of suitable restriction sites, and a high copy number potential. Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses. Expression vectors that provide suitable levels of expression of a polypeptide in different hosts are well known in the art. Bacterial expression vectors well known in the art include pET11a (Novagen), λ gt11 (Invitrogen).

The invention further relates to the use of a nucleic acid, expression cassette or vector of the invention for transforming, transfecting or transducing a host cell. The choice of the vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced.

The invention also relates to a host cell or recombinant cell comprising a nucleic acid, cassette or vector of the invention. The host cell may be transformed, transfected or transduced in a transient or stable manner. The expression cassette or vector of the invention is introduced into a host cell so that the cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The expression cassette or vector can be introduced into the host cell using standard techniques. Examples of such techniques include transformation, transfection, lipofection, protoplast fusion, and electroporation.

The host cell may be any cell that can be genetically modified, and is preferably cultured. The cell may be eukaryotic or prokaryotic, such as a mammalian cell, an insect cell, a plant cell, a microorganism such as a yeast, a fungal or bacterial cell, or the like. In a particular embodiment, the host cell is selected from the group consisting of Escherichia Coli (Escherichia Coli), Bacillus (Bacillus), Lactobacillus (Lactobacillus), Streptomyces (Streptomyces), Trichoderma (Trichoderma), Aspergillus (Aspergillus), Pichia (Pichia) or Yarrowia (Yarrowia). It will be appreciated that the invention is not limited with respect to any particular cell type and can be applied to all kinds of cells following common general knowledge. The term "host cell" also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication.

In a particular embodiment, the host cell is a yeast, preferably yarrowia, and the polypeptide produced is a glycosylated polypeptide that exhibits greater thermostability. In a preferred embodiment, the polypeptide consists of SEQ ID No. 1 comprising at least one glycosylation of an amino acid residue selected from N28, N158 or N165.

In a particular embodiment, the invention provides a host cell engineered to express a nucleic acid as set forth in SEQ ID NO. 2 or an expression cassette thereof.

In a particular embodiment, the invention provides a recombinant Bacillus subtilis engineered to express a nucleic acid as set forth in SEQ ID NO. 2 or an expression cassette thereof.

In another specific embodiment, the invention provides a recombinant E.coli engineered to express a nucleic acid as set forth in SEQ ID NO. 2 or an expression cassette thereof.

In another specific embodiment, the invention provides a recombinant Yarrowia lipolytica (Yarrowia lipolytica) engineered to express the nucleic acid shown in SEQ ID NO:2 or an expression cassette thereof.

In another embodiment, the invention provides a host cell comprising and expressing a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 94%, 95%, 99% or 100% identity to the full length amino acid sequence set forth in SEQ ID No. 1 and having polyester degrading activity.

In another embodiment, the invention provides a host cell comprising and expressing a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 94%, 95%, 99% or 100% identity to the full length amino acid sequence as set forth in SEQ ID No. 1 and having PLA degrading activity, more preferably PLLA degrading activity.

In a particular embodiment, the host cell is a recombinant microorganism. The present invention actually allows the engineering of microorganisms with improved ability to degrade polyester-containing materials. For example, the sequences of the invention can be used to supplement wild-type strains of fungi or bacteria that have been described as capable of degrading polyesters to improve and/or increase strain capacity.

In a particular embodiment, the present invention provides a recombinant bacillus subtilis comprising and expressing a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full-length amino acid sequence as set forth in SEQ ID No. 1 and having polyester degrading activity, preferably PLA degrading activity, more preferably PLLA degrading activity.

In another specific embodiment, the invention provides a recombinant E.coli comprising and expressing a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the full length amino acid sequence as set forth in SEQ ID NO. 1 and having polyester degrading activity, preferably PLA degrading activity, more preferably PLLA degrading activity.

In another specific embodiment, the invention provides a recombinant yarrowia lipolytica comprising and expressing a nucleotide sequence encoding a polypeptide comprising an amino acid sequence at least 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the full-length amino acid sequence set forth in SEQ ID No. 1 and having polyester degrading activity, preferably PLA degrading activity, more preferably PLLA degrading activity. Advantageously, one or several amino acid residues of the polypeptide are glycosylated. More preferably, the polypeptide consists of SEQ ID NO 1 comprising at least one glycosylation of an amino acid residue selected from N28, N158 or N165, preferably at least N158.

It is another object of the invention to provide a method for producing a polypeptide of the invention comprising (i) culturing a recombinant cell as defined above, (ii) recovering the culture supernatant, and optionally (iii) isolating or purifying the polypeptide. The invention further relates to said polypeptide obtained by this production method.

Alternatively, the polypeptides of the invention may be produced by Cell-Free Methods (Kim et al, Jbiosci. Bioeng. July 2009; Spirin et al (2007) Front Matter in Cell-Free Protein Synthesis: Methods and Protocols), or may be chemically synthesized.

Degradation of polyester-containing materials

The invention provides methods of using the polypeptides of the invention to degrade and/or recycle polyester-containing materials, such as plastic articles made from or containing polyester, under aerobic or anaerobic conditions. Indeed, due to its high polyester depolymerization efficiency, the polypeptide of the invention is more advantageous than other known chemical or microbial polyester degradation means. The polypeptides of the invention have an increased rate of depolymerization, particularly for PLA depolymerization.

It is therefore an object of the present invention to use the polypeptides of the present invention or the corresponding recombinant cells or extracts or compositions thereof for the enzymatic degradation of polyester-containing materials. In a preferred embodiment, the polypeptide or the corresponding recombinant cell, extract or composition thereof is used for the enzymatic degradation of PLA-containing material, and more preferably for the enzymatic degradation of PLLA-containing material.

It is another object of the present invention to degrade polyester-containing materials using polypeptides comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the full-length amino acid sequence as set forth in SEQ ID No. 1 and having polyester degrading activity.

It is another object of the present invention to provide a method for degrading a polyester containing material, wherein the polyester containing material is contacted with a polypeptide of the present invention or a corresponding recombinant cell or an extract or composition thereof. Advantageously, the polyester containing polyester material is depolymerized until it becomes monomeric and/or oligomeric. In a particular embodiment, all of the target polyester is depolymerized until it becomes a monomer of the original polyester forming the material.

In one embodiment of the degradation process, at least one polyester is degraded to produce a repolymerizable monomer and/or oligomer, which is advantageously recovered for reuse.

In another embodiment, the polyester containing polyester material is completely degraded.

In a preferred embodiment, the polyester containing material comprises PLA, more preferably PLLA, and at least lactic acid monomers and/or oligomers are recovered, e.g. for recycling or methanation.

In another embodiment, the polyester-containing material comprises PLA and at least one additional polyester preferably selected from the group consisting of: polytrimethylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polybutylene adipate-co-terephthalate (PBAT), Polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), Polycaprolactone (PCL), poly (ethylene adipate) (PEA) and blends/mixtures of these polyesters.

Alternatively or in addition, the polyester-containing material may further contain at least one polyamide (also known as nylon) and/or at least one polyolefin preferably selected from Polyethylene (PE), polypropylene (PP) and blends/mixtures of these polymers, and/or at least one vinyl polymer made from vinyl monomers, i.e. small molecules containing carbon-carbon double bonds.

Alternatively or in addition, the polyester-containing material may further comprise at least one natural polymer (i.e. not petrochemical-derived), preferably selected from starch, flour, cellulose and blends/mixtures thereof.

In a particular embodiment, the polyester-containing material comprises Polyhydroxyalkanoate (PHA) and/or polyethylene terephthalate (PET) and/or polybutylene adipate-co-terephthalate (PBAT) or blends/mixtures of these polyesters.

According to the invention, the polyester-containing material may also contain metal compounds, inorganic compounds, glass compounds, natural or synthetic fibers, paper, wood compounds like lignin, cellulose or hemicellulose, starch and derivatives thereof.

The invention also relates to a method of producing monomers and/or oligomers from a polyester containing material comprising exposing the polyester containing material to a polypeptide of the invention or a corresponding recombinant cell or an extract or composition thereof, and optionally recovering the monomers and/or oligomers. The process of the present invention is particularly useful for the production of lactic acid monomers.

When using recombinant microorganisms, said microorganisms advantageously exhibit a modified metabolism to prevent the consumption of monomers and/or oligomers obtained from degraded polyesters. For example, enzymes in the microorganism that degrade the monomers and/or oligomers have been deleted or knocked out. Alternatively, the methods of the invention can be performed in a medium containing at least one carbon source that can be used by a recombinant microorganism such that the microorganism preferentially consumes this carbon source over monomers and/or oligomers. Advantageously, the polyester-containing material is contacted with a medium containing the recombinant microorganism, glucose or the like as a carbon source, and an available nitrogen source, including organic nitrogen sources (e.g., peptone, meat extract, yeast extract, corn steep liquor) or inorganic nitrogen sources (e.g., ammonium sulfate, ammonium chloride). If necessary, the medium can further contain inorganic salts (such as sodium ion, potassium ion, calcium ion, magnesium ion, sulfate ion, chloride ion, phosphate ion). In addition, the medium may also be supplemented with trace components such as vitamins, minor elements and amino acids.

In a particular embodiment, the polyester-containing material may be pre-treated prior to contact with the polyesterase of the invention to physically alter its structure so as to increase the contact surface between the polyester and the polyesterase. For example, the polyester-containing material may be converted to an emulsion or powder, which is added to a liquid medium containing the polypeptide of the invention and/or the recombinant microorganism or extract thereof. Alternatively, the polyester-containing material may be mechanically ground, pelletized, etc. by cutting, impacting, crushing, grinding, segmenting, cryomilling, etc. to reduce the shape and size of the material prior to addition to the liquid medium containing the recombinant microorganism, extract thereof, and/or polypeptide. Mechanical pretreatment may also be sonication, centrifugation, shearing, impact, use of a high pressure homogenizer, dipping or liquefaction with a rotating drum, use of a screw press, disc screen mill or piston press. Alternatively or additionally, thermal pre-treatment may be applied. This can be achieved with microwaves. The thermal pretreatment may provide sterilization, pasteurization, or sterilization. In another embodiment, the polyester-containing material is chemically pretreated to improve its structure and increase the contact surface between the polyester and the polypeptide of the invention. Bases, acids, solvents or ionic liquids may be used. Ozonation may also be performed. In a particular embodiment, the polyester-containing material may also be sorted, washed, disinfected, sterilized, and/or biologically cleaned prior to degradation. According to the invention, several intervention processes can be combined.

The time required to degrade the polyester-containing material may vary depending on the polyester-containing material itself (i.e., the nature and source of the plastic article, its composition, shape, etc.), the type and amount of polypeptide used, and various process parameters (i.e., temperature, pH, additional reagents, etc.). The process parameters can be readily adapted to the polyester-containing material by those skilled in the art.

Advantageously, the process is carried out at a temperature comprised between 20 ℃ and 90 ℃, preferably between 20 ℃ and 60 ℃, more preferably between 30 ℃ and 55 ℃, more preferably between 40 ℃ and 50 ℃, even more preferably at 45 ℃. More typically, the temperature is maintained below an inactivation temperature, which corresponds to the temperature at which the polypeptide is inactivated and/or the recombinant microorganism no longer synthesizes the polypeptide.

The pH of the medium may be in the pH range of 5-11, preferably in the pH range of 7-10, more preferably in the pH range of 8.5-9.5, even more preferably in the pH range of 8-9. Advantageously, the pH is adjusted according to the solubility of the target polyester and the target monomer/oligomer to improve process efficiency. Preferably, the pH is adjusted to maintain an optimal pH for the polypeptide. In effect, depolymerization of the polyester produces acidic monomers and oligomers that induce a decrease in pH. Addition of a dilute or saturated base such as calcium hydroxide can be used to counteract this acidification and maintain the pH at an optimal pH.

Advantageously, the polypeptide is added in an amount in the range of 0.001% to 5%, preferably in the range of 0.001% to 1%, more preferably in the range of 0.001% to 0.1%, even more preferably in the range of 0.001% to 0.05% by weight of the polyester containing material.

In a particular embodiment, the method is performed under agitation, preferably comprised between 30rpm and 2000rpm, to facilitate contact between the polypeptide and the polyester containing material.

In a particular embodiment, at least a lipophilic and/or hydrophilic agent is added to the medium to improve the depolymerization step. An inducer such as an oligomer of the polyester or derivative thereof can be added to the culture medium comprising the recombinant microorganism to improve polypeptide production. Surfactants such as Tween (Tween) or small proteins such as hydrophobin (hydrophibin) may be added to the medium to improve the interfacial energy between the polyester and the polypeptide or recombinant microorganism and to improve the efficiency of degradation. Organic substances or ionic liquids can be used to swell the polyester and increase its accessibility by microorganisms or polypeptides.

The reaction time for depolymerising the at least one polyester of the plastic material until it becomes monomeric/oligomeric is advantageously comprised between 5 hours or less and 110 hours, more preferably between 24 hours and 72 hours. The reaction time may allow the depolymerization to progress sufficiently and will not be economically disadvantageous. The reaction time may be longer for anaerobic biodegradation in methanation sites, or for aerobic biodegradation in the natural environment.

Optionally, monomers and/or oligomers resulting from depolymerization may be recovered sequentially or continuously. Depending on the starting polyester-containing material, a single type of monomer and/or oligomer or several different types of monomers and/or oligomers may be recovered.

The recovered monomers and/or oligomers can be further purified using all suitable purification methods and conditioned to a repolymerizable form. Examples of purification methods include stripping methods, separation by aqueous solutions, steam selective condensation, filtration and concentration of media after biological processes, separation, distillation, vacuum evaporation, extraction, electrodialysis, adsorption, ion exchange, precipitation, crystallization, concentration and acid addition dehydration and precipitation, nanofiltration, acid catalyst treatment, semi-continuous or continuous distillation, solvent extraction, evaporative concentration, evaporative crystallization, liquid/liquid extraction, hydrogenation, azeotropic distillation methods, adsorption, column chromatography, simple vacuum distillation, and microfiltration, which purification methods may or may not be combined.

The repolymerizable monomers and/or oligomers can then be used again, for example, to synthesize a polyester. Advantageously, polyesters having the same properties are repolymerized. However, it is possible to mix the recovered monomers and/or oligomers with other monomers and/or oligomers, for example to synthesize new copolymers. Alternatively, the recovered monomer can be used as a chemical intermediate to produce a new target compound.

In a particular embodiment, the repolymerization is carried out using a hydrolase under conditions suitable to allow the polymerization reaction to proceed. An initiator may be added to the monomer/oligomer solution to facilitate polymerization. The person skilled in the art can easily adapt the process parameters to the monomers/oligomers and to the polymer to be synthesized.

In a particular embodiment, the process of the invention is carried out in a reactor. "reactor" designates any device or equipment or facility suitable for maintaining and converting plastic articles. The reactor may comprise inlet and outlet means to supply/collect media, nutrients, gases etc. The reactor may be closed or open, such as a tank.

Plastic compound and article

It is another object of the present invention to provide a polyester-containing material comprising the polypeptide of the present invention and/or a recombinant microorganism expressing and secreting the polypeptide. In a particular embodiment, the polyester-containing material may be a plastic compound.

It is therefore an object of the present invention to provide a plastics compound comprising a polypeptide and/or recombinant cell of the invention and/or a composition or extract thereof; and at least one polyester. In a preferred embodiment, the polyester is selected from polylactic acid, preferably from PLLA. In particular, the plastic compound may contain additional polymers preferably selected from the group consisting of: polyesters such as PDLA, PBAT, PHA, PCL, PET; polyolefins such as polyethylene, polypropylene; or natural polymers such as starch, cellulose or flour; and blends/mixtures thereof. More particularly, the plastic compound may contain an additional polymer selected from PBAT and flour or starch.

In a particular embodiment, the polypeptide used to prepare the plastics compound comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 92%, 95%, 99% or 100% identical to the full length amino acid sequence set forth in SEQ ID No. 1.

In particular, the invention relates to a method comprising the steps of: mixing the polyester and the polypeptide degrading the polyester and/or the recombinant cell of the invention at a temperature at which the polyester is in a partially or fully molten state such that the polypeptide and/or the recombinant cell is integrated into the exact structure of the polyester-containing material. In a particular embodiment, spores of the recombinant microorganism are included into the polyester-containing material.

For example, the polypeptide and/or recombinant microorganism of the invention and the polyester can be mixed at a temperature between the glass transition temperature and the melting point of the polyester. Alternatively, the biological entity and the polyolefin may be mixed at a temperature corresponding to the melting point of the polyester or above. In a particular embodiment, the polypeptide/microorganism and the polyester are mixed at a temperature between 80 ℃ and 250 ℃, preferably between 100 ℃ and 200 ℃. Alternatively, the polypeptide/microorganism and the polyester are mixed at a temperature above 80 ℃, preferably above 100 ℃, even more preferably above 130 ℃. More generally, the polypeptide and/or recombinant microorganism advantageously is at least resistant to the extrusion temperature of the polyester.

More preferably, the mixing step is performed using extrusion, twin screw extrusion, single screw extrusion, injection molding, casting, thermoforming, rotational molding, pressing, calendering, flattening, coating, layering, expanding, pultrusion, extrusion blow-molding, extrusion-expansion, press-granulation, water-in-oil-in-water double emulsion evaporation, or any technique known to those skilled in the art.

The resulting plastic compound incorporates the polypeptide/microorganism of the invention embedded in the bulk of the compound.

Advantageously, the plastic compounds can be used for the manufacture of plastic articles also comprising the polypeptides/microorganisms of the invention therein.

In a particular embodiment, the resulting plastic compound or plastic article is a biodegradable plastic compound or plastic article that meets at least one relevant standard and/or label known to those skilled in the art, such as standard EN 13432, standard ASTM D6400, good biodegradable Soil (OK Biodegradation Soil oil,

label), good biodegradable Water (OK Biodegradation Water,

label), good Compost (OK composite,

label), good Home Compost (OK composite Home,

a label).

By biodegradable plastic compound or plastic article is meant a plastic compound or plastic article that is at least partially converted to water, carbon dioxide or methane and biomass under ambient conditions. As illustrated in the examples, the preferred plastic compounds or plastic articles of the present invention are biodegradable in water. Preferably, about 90% by weight of the plastic compound or plastic article is biodegraded in water in less than 90 days, more preferably in less than 60 days, even more preferably in less than 30 days. Alternatively or additionally, the plastic compound or plastic article may be biodegradable upon exposure to humid and temperature conditions present in a landscape. Preferably, about 90% by weight of the plastic compound or plastic item is biodegraded in the environment in less than 3 years, more preferably in less than 2 years, even more preferably in less than 1 year. Alternatively, plastic compounds or plastic articles can be biodegraded under industrial composting conditions, wherein the temperature is maintained above 50 ℃.

Other aspects and advantages of the present invention will be disclosed in the following examples, which should be considered illustrative and not limiting the scope of the present application.

Examples

Example 1: purification and characterization of the polyesterase from actinomadura keratinocyte T16-1

The keratin-degrading actinomadura strain NBRC 104111 strain T16-1(Sukkum et al 2009) isolated from thailand forest soil was selected because its supernatant had high PLA-degrading activity.

Production of enzymes in fermentors

In a 10L fermenter: (

Biostat Cplus) was performed. 500mL of yeast malt broth (YM, Sigma-Aldrich) preculture was used to inoculate 4.5L of basal medium (2.4g/L gelatin; 4g/L (NH)₄)₂SO₄；0.2g/L MgSO₄.7H₂O; 0.5g/L yeast extract; 4g/L K₂HPO₄；2g/L KH₂PO₄Adjusted with NaOH at pH 6.8). The temperature was regulated at 46 ℃ and 10% (v/v) H was added₃PO₄The solution maintained the pH at 6.8. The stirring rate was fixed at 70rpm to enable gentle mixing and the aeration rate (0.6 to 1.6vvm) was adjusted to provide a dissolved oxygen level in the reactor above 20% of air saturation to avoid any oxygen limitation in the culture. The fermentor was connected to a computer and the MFCS/DA software was tuned to the controlled parameters (pH, temperature, partial pressure of dissolved oxygen, and H)₃PO₄Add) to perform online acquisition and allow online monitoring and regulation of these parameters.

The duration of the incubation was 50 hours. The supernatant containing the extracellular enzyme was recovered by centrifugation (13000 g-10 min) and stored at 4 ℃.

Purification of polyesterase

The supernatant of the culture was concentrated 40-fold using a 500mL Amicon cell (Merck Millipore) and cellulose regeneration membrane (GE Healthcare Life Science) with a pore size of 10 KDa. The resulting solution was dialyzed against 50mM glycine-NaOH pH 10 buffer.

AKTA purifier device (GE Healthcare Life Science) was used for polypeptide purification using an anion exchange purification HiTrap Q FF 1mL column (GE Healthcare Life Science) with 50mM glycine-NaOH (pH 10) as loading buffer. Elution was performed with 50mM glycine-NaOH pH 10 buffer containing a gradient of 0 to 1M NaCl.

The presence of the polyesterase of the invention in the different fractions including the flow-through fraction was demonstrated by stain-free SDS-PAGE gels (Bio-Rad) after TCA precipitation (v/v) and by testing the ability of the enzyme to produce halos in plates containing a mixture of agarose (1%) and PLLA emulsion (0.5%, Natureplast).

Determination of the N-terminal sequence of the polypeptide

After passive extraction from the gel, the polypeptides contained in the desired bands were N-terminally sequenced by Edman microsequencing technique using a 494 microsequencer device (Perkin Elmer Applied Biosystems) in the Pissaro platform (France) of Rouen.

Molecular biology techniques

Unless otherwise specified, the general procedure for DNA manipulation is as previously described (Sambrook J, Russell DW.2001.molecular cloning: a Laboratory Manual, 3 rd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs and used according to the manufacturer's instructions. PCR was performed using the CloneAmp HiFi PCR premix (Takara-Clontech). Synthetic oligonucleotides were synthesized by Eurogentec. The PCR product was purified using GenElute PCR purification kit (Sigma-Aldrich). Plasmid DNA was introduced into E.coli strain DH5 alpha (Invitrogen) using a heat shock method. Obtained from E.coli using QIAprep spin plasmid Mini prep kit (Qiagen)Plasmid DNA. Use of

Enhanced mini kit (Qiagen) to obtain total RNA samples, and subsequently, using Ribo-Zero^TMThe rRNA removal kit (Epicentre) depletes ribosomal RNA.

RNA sequencing

Two RNA libraries corresponding to the expression profile of the keratin-degrading actinomadura strain T16-1 in the presence/absence of PLLA (NaturePlast, 500 μm) were constructed using the Illumina TruSeq chain mRNA kit and the ultra high throughput sequencing System Ilumina HiSeq 2500. Paired-end reads (2x100 ob) were obtained using chemistry v3 of TruSeq SBS kit (illumina).

Bioinformatics

Using TBLASTN, BLASTX and BLASTP (Altschul SF et al 1997 Gapped BLAST and PSI-BLAST: a new generation of polypeptide database search programs. nucleic Acids Res.25: 3389-3402), the National Center for Biotechnology Information (website) website (available at National Center for Biotechnology Information) was usedhttp://www.ncbi.nlm.nih.gov) The accessed non-redundant sequence database performs a database search. Sequence analysis was performed using Vector NTI software (Life Technologies) and multiple local alignments were performed using ClustalW software (Thompson JD, Higgins DG, Gibson TJ.1994.CLUSTAL W: optimizing the sensitivity of progressive multiple sequence alignment through sequence alignment, position-specific gap polarities and weight matrix choice. nucleic acids. Res.22: 4673-.

Results

Polypeptide purification

Purification of the polypeptide was achieved at pH 10 using an anion exchange column. The activity of the different fractions was tested on PLA-containing agarose plates. Halo formation indicating PLA hydrolysis was only obtained in case of flow through fractions. SDS-PAGE performed in the case of beta-mercaptoethanol showed that the flow-through fraction contained distinct bands (FIG. 1). This purification process enables to obtain a pure polypeptide exhibiting the PLA hydrolytic activity to be obtained. The molecular weight of the polypeptide was estimated to be 27 kDa.

N-terminal sequencing of the mature protein contained in the band revealed a unique 28 amino acid sequence:

ATQNNPPSWGLDRIDQTNLPLSRSYTYN(SEQ ID NO:3)

the polypeptide purified in the flow through shows disaggregation activity towards PLA powder. The specific activity of the polypeptide was 4.8g lactic acid produced per mg of enzyme and per hour (according to the protocol below).

RNA sequencing results allowed the identification of DNA sequences encoding polypeptides whose sequences showed 100% identity to the 28 amino acids previously identified by N-terminal sequencing.

The determined DNA sequence (SEQ ID NO:4) is reproduced below, with the underlined sequences corresponding to the hypothetical peptide signal and the propeptide:

5’atgagacgacgtaccctgcccatcgccgtcctcgccgccgttcccctggccgtggcgggcgccctg cccgccggagccgcccccgccgcccccgccgtcccggtcgccatggcggccgccggacagggcgtcgccggacagt acatcgtgacgctgaagaagggcgtctcggtcgactcgaccgtcgccaagcgcggaatccgcacccagcaccgttt cggcaaggtgctgaacggcttctccgccaagctcaccgatgaccaactgtccaagctgcgcaccacgcccggtgtc gcgtccatcgagcaggacgccgtcatcacggtggacgccacgcagaacaacccgccgtcgtggggcctggaccgcatcgaccagacgaacctgccgctgtcgcgcagctacacctacaattccaccggcgcgggcgtgaacgcctacatcatcgacaccggcatctacaccgcgcactccgacttcggcggccgcgccaccaacgtctacgacgccctcggcggcaacggccaggactgcaacggccacggcacccacgtcgcgggcaccgtcggcggcgccgcctacggcgtggccaaggcggtcaacctgcgcggcgtgcgcgtgctcaactgcagcggcagcggcaccacctccggtgtcatcgccggcatgaactgggtggccagcaaccacgtcaagcccgccgtggcgaacatgtcgctgggcggcggctactcctcctccctgaacacggccgccaacaacctggccagctccggcgtgttcctggccgtcgccgcgggcaacgagaccaccaacgcctgcaaccgctcgccggccagcgccgccaacgccaccacggtcgccgcgagcaccagcaccgacgcccgggcctcctacagcaactacggctcgtgcgtccacctgtacgcgcccggctcgtccatcacctccgcctggctgaacggcggcaccaacaccatcagcggcacgtcgatggccacgccgcacgtggccgggaccgccgccctctacaaggcgacctacggcgacgcctcgttcagcaccatccgcagctggctggtcagcaacgccacctccggcgtcatcaccggcaacgtgtcgggcaccccgaacctgctgctgaacaagcgctccctgtaa 3’(SEQ ID NO:4)

the encoded polypeptide exhibits a sequence of 386 amino acids (SEQ ID NO:5 below), where

Residues 1 to 29 correspond to the peptide signal,

residues 30 to 110 correspond to the hypothetical propeptide, and

residues 111 to 386 correspond to the mature polypeptide.

SEQ ID NO:5 (underlined propeptide sequence; double underlined peptide signal):

mature polypeptide sequence (SEQ ID NO: 1):

ATQNNPPSWGLDRIDQTNLPLSRSYTYNSTGAGVNAYIIDTGIYTAHSDFGGRATNVYDALGGNGQDCNGHGTHVAGTVGGAAYGVAKAVNLRGVRVLNCSGSGTTSGVIAGMNWVASNHVKPAVANMSLGGGYSSSLNTAANNLASSGVFLAVAAGNETTNACNRSPASAANATTVAASTSTDARASYSNYGSCVHLYAPGSSITSAWLNGGTNTISGTSMATPHVAGTAALYKATYGDASFSTIRSWLVSNATSGVITGNVSGTPNLLLNKRSL

the theoretical molecular weight calculated for this 276 amino acid mature polypeptide is 27.7kDa, which corresponds to the molecular weight observed after purification of the polyesterase from the supernatant of the keratin degrading actinomadura T16-1.

Based on homology to known polypeptides, a polypeptide with polyesterase activity revealed the presence of two putative calcium binding sites, with residues Ala 172, Ala 174 and His 197 in the first position and residues Asp 12, Asp 15 and Gln 16 in the second position. The catalytic site of the polypeptide consists of the amino acids His 71, Asp 40 and Ser 221. In addition, two putative disulfide bonds between residues Cys 68-Cys 100 and Cys 164-Cys 195 have also been identified in the polypeptide sequence (fig. 2).

Example 2: the polyesterase from actinomadura keratindegrading strain T16-1 was characterized.

The optimal pH and temperature of the enzyme was determined and the thermostability of the enzyme was investigated.

Optimal pH and temperature of the polypeptide

The optimum pH of the enzyme is 8.5. The depolymerization test was performed at 50 ℃ in a pH range between 7 and 9 with 2mL buffer containing 600. mu.g enzyme and 20mg Goodfellow PLA film in a magnetically stirred tube. At pH 7, the enzyme showed some activity.

Depolymerization tests were performed at 37 deg.C, 45 deg.C and 50 deg.C, at pH8.5, with 2mL of pH8.5Tris-HCl buffer containing 600. mu.g of enzyme, and 20mg of Goodfellow PLA film. The optimum temperature for the enzyme is 50 ℃.

Polyesterase thermal stability

The determination of the stability of the polyesterase was carried out at a temperature ranging from 4 ℃ to 60 ℃. The polyesterase remained stable for several months at 4 ℃. The compromise between stability and activity of the enzyme corresponds to a temperature of 45 ℃. The enzyme half-life was 2.5 weeks at 45 ℃. Furthermore, there was no loss of polyester degradation activity after the lyophilization procedure.

Example 3: PLA degradation methods were developed.

Given that lactic acid is a potential inhibitor and that the pH is drastically reduced during the reaction, the aim of these experiments was to solve the problem of lactic acid production. Enzymes and PLA were introduced and confined in a dialysis tube of 10 kDa. This tube is permeable to lactic acid and is placed in a volume of buffer allowing operation at constant pH and diluting the lactic acid, thereby limiting its potential inhibitory effect.

Enzymatic PLA degradation

The degradation capacity of the polypeptide of interest (SEQ ID NO:1) was studied during the hydrolysis kinetic analysis of PLA. Conversion of PLA to lactic acid was followed by HPLC analysis.

PLA degradation assays were performed in 10kDa dialysis tubes (cellulose membrane, 25mm width, Sigma-Aldrich D9777-100 FT). 3mL of 100mM Tris HCl pH8.5 buffer, PLLA powder (Natureplast 500 μm), PLA film (Goodfellow, 50 μm thickness) or PLA pieces containing 90 μ g of enzyme were introduced and confined in dialysis tubing. The tubes were placed in 50mL (unless otherwise specified) of 100mM Tris-HCl buffer to control the pH to 8.5. The buffer was supplemented with kanamycin (40. mu.g/mL) to avoid any contamination. The reaction was incubated at 45 ℃ with stirring (150 rpm). The goal of this procedure is to control the pH of the reaction and avoid potential enzyme inhibition by lactic acid.

HPLC analysis was carried out by buffering the outside of the tube (column: Aminex HPX-87H (300 mM. times.7.8 mM), mobile phase: 5mM H₂SO₄Temperature: 50 ℃, flow rate: 0.5mL.min^-1Injection volume: 20 μ L) to quantify the degradation products (lactic acid and soluble oligomers). Standards of lactic acid (Sigma-Aldrich L1750-10G), dimer, and trimer (made by house) were used for external calibration.

To quantify the benefits of this approach, PLA hydrolysis was achieved with the following dialysis system: in a magnetically stirred tube at 45 ℃ containing a 50mg PLA film (Goodfellow, 50 μm thickness, 2% D-lactic acid), 3mL of 100mM Tris-HCl pH8.5 buffer containing 90 μ g of enzyme, was introduced into the tube. After 24 hours of reaction, 55% conversion was obtained in the proposed reactor.

Example 4: polyesterase activity was evaluated on PLA/PLLA with different granulometry values.

Enzyme activity was assessed during hydrolysis of PLA powder (PLLA NaturePlast, PLA Ingeo7001D containing 4% D-lactic acid). Different particle sizes (100-.

Table 1:PLA (Ingeo 7001D) and plla (natureplast) powders: particle size and crystallinity.

	Particle size	Tg(℃)	Degree of crystallinity (%)
				PLA Ingeo 7001D	100-250μm	62	3
	250-500μm	60	7
					500μm-1mm	63	41
	1mm-2mm	62	42
				PLLA Natureplast	100-250μm	58	23
	250-500μm	57	24
					500μm-1mm	59	34
	1mm-2mm	64	43

Differential Scanning Calorimetry (DSC) test is used to determine the glass temperature (Tg) and crystallinity of PLA for approximately 8mg of sample using a Q100 TA-RCS 90 instrument in an aluminum pan under a nitrogen atmosphere (50mL/min) at a scan rate of 10 deg.C/min from-50 deg.C to 300 deg.C.

In the reactor procedure described in example 3, the hydrolysis performance of two different PLA powders with different particle sizes was determined with 100mg PLA powder, 2mL 100mM Tris-HCl pH8.5 buffer containing 60. mu.g enzyme. The hydrolysis of PLA and PLLA powders with the same size was the same, indicating that the presence of 4% D-lactic acid was not detrimental to the hydrolysis performance. PLA crystallinity in the range 5 to 24% has a lower impact on hydrolysis performance. Conversely, there is a strong influence of particle size on the hydrolysis rate of PLA and PLLA powders: the finer the powder, the more efficient the hydrolysis rate. This can be explained by the increase in the exchange surface between the solid and liquid phases. (FIGS. 3 and 4).

Particle sizes in the range of 100-250 μm enable a conversion of 68% to be obtained in 24 hours.

If 10mM CaCl is added to the reactor₂Then 95% conversion of 500 μm PLLA powder NaturePlast is obtained after 80 hours of reaction.

Example 5: effect of PLA concentration on enzymatic Activity

The enzyme activity was assessed at different concentrations (33 to 300g/L) during hydrolysis of PLLA powder using the same protocol as described in example 3, 3mL of 100mM Tris-HCl pH8.5 buffer containing 90. mu.g of enzyme. The results are presented in table 2.

The higher the PLLA concentration, the higher the productivity of lactic acid formation, which tends to reach 0.2g lactic acid/mg enzyme/h in the case of PLLA at a concentration of 300 g/L.

Table 2:productivity obtained during the reaction for 10 hours during the polyesterase catalyzed hydrolysis of different concentrations of PLLA.

Productivity 10h	PLLA 33g/L	PLLA 100g/L	PLLA 200g/L	PLLA 300g/L
					g lactic acid/mg enzyme/h	0.07	0.15	0.17	0.19
Improvement factor	1	x2.1	x2.3	x2.6

Example 6: polyesterases catalyze the hydrolysis of PLA films to lactic acid.

The same experimental protocol presented in example 3 was used during hydrolysis of PLA films (Goodfellow, 50 μm thickness, 2% D-lactic acid). Kinetic analysis was performed at 45 ℃ and pH8.5 using 3mL100mM Tris-HCl pH8.5 buffer containing 90. mu.g of the polyesterase and 50mg of membrane (17 g/L).

The polyesterase was able to hydrolyze the film to lactic acid. A conversion of 76% was obtained within 48 hours and a conversion of 82% was obtained within 72 hours (fig. 5).

Example 7: polyesterases catalyze the hydrolysis of PLA commercial articles to lactic acid.

The same experimental protocol presented in example 3 was used during the polyesterase catalyzed hydrolysis of commercial articles (PLA cups, plates, films and tableware). The powders (250-500 μm) of these articles were subjected to hydrolysis tests. 100mg of commercial powder, 3mL of 100mM Tris-HCl pH8.5 buffer containing 90. mu.g of enzyme were used.

The initial rate of hydrolysis was relatively similar for whatever PLA article (from 27% for films to 44% for cups at 10 hours). This is in the same range compared to the results obtained with pllannatureplast powder (37%). However, it is noteworthy that PLA cups are more easily converted to lactic acid than PLLA powder. 98% conversion of PLA cups was obtained after 48 hours. Over 72 hours, 93% and 84% conversion to lactic acid was obtained for the film and the tray, respectively. Tableware is the most difficult item to degrade, with up to 60% of the items being converted to lactic acid. It contains about 1% TiO₂And shows TiO₂The presence of (b) does not lead to this phenomenon.

The polyesterase was able to hydrolyze all commercial articles to lactic acid (fig. 6).

Example 8: recycling method Using the enzyme of SEQ ID NO 1

The purpose of these experiments was to verify the industrial applicability of the PLA degradation solution of the present invention, wherein the enzyme of SEQ ID NO:1 and PLA were introduced into a reactor without any dialysis system.

PLA degradation assays were performed directly with enzyme production media obtained by fermentation as described in example 1. The supernatant containing the extracellular enzyme was recovered by centrifugation (13000 g-10 min) and stored at 4 ℃.

400mg of PLA Natureplast powder (particle size <500 μm) was added directly to 25ml of supernatant. 300mg calcium carbonate and 100mg calcium hydroxide were added to neutralize the lactic acid released during hydrolysis and maintain the pH of the solution above 7. At the beginning of the reaction, the pH was between 9.0 and 9.8. The reaction mixture was incubated at 45 ℃ for 140 hours with stirring (300 rpm).

During the reaction, several samples of the mixture (1ml) were collected and filtered on a 0.22 μm filter. 20 μ l of the filtrate was analyzed by HPLC as described in example 2 to quantify the lactic acid and soluble oligomers (DP 2). After 144 hours of reaction, 17.5g/l lactic acid and 0.52g/l DP2 oligomer were obtained. The conversion yield of PLA to lactic acid was greater than 77% (fig. 7).

Example 9: comparison of PLA degradation by different specific polypeptides of the invention

The amino acid sequence SEQ ID NO 1 has been modified to improve its thermostability.

The first strategy consists in making two amino acid residue substitutions in the amino acid residue sequence SEQ ID NO:1 by introducing two cysteine residues (T175C and R247C) at residue positions 175 and 247 of SEQ ID NO:1 to introduce additional disulfide bonds into the structure of the polypeptide.

The second strategy consists in introducing an additional salt bridge between amino acid residues 139 and 170 or between amino acid residues 143 and 173 of SEQ ID NO. 1. Thus, the first resulting variant contains the amino acid residue substitutions N139D and S170R, and the second resulting variant contains the amino acid residue substitutions N143R and N173E.

The third strategy consisted in site-directed mutagenesis of the nucleic acid sequence shown as SEQ ID NO. 2 to generate 5 variants, each containing an amino acid residue substitution selected from S194P, H197D, L210P, G212N and I217K.

Other variants of SEQ ID NO:1, each containing substitutions of amino acid residues selected from R166K, T160A and L138A, have been tested and have been measured to be comparable in activity to the native polypeptide of SEQ ID NO: 1.

Example 10: recombinant expression and purification of the polyesterase of SEQ ID NO 1.

The polyesterase of SEQ ID NO 1 was expressed in three different hosts: yarrowia lipolytica, bacillus subtilis, and escherichia coli.

10A-recombinant expression of polyesterase in yarrowia lipolytica

Expression of the polyesterase of SEQ ID NO:1 under the control of the constitutive promoter TEF in the yeast Yarrowia lipolytica, more precisely in strain JMY1212, as previously described by Bordes et al, 2007(F. Bordes, F. Fudalej, V.Dossat, J.M.Nicaud, et A.Marty (2007) A new recombinant protein expression system for high-through high expression screening in the yeast Yarrowia polylithica.J.of Mibrob.meth.,70,3,493 502). The sequence of the propeptide corresponding to the sequence of the gene followed by expression of the mature polyesterase was optimized for the codon usage of yarrowia lipolytica. This sequence was integrated downstream of the secretion signal sequence of the gene encoding lipase lip2 from yarrowia lipolytica.

Then in the presence of medium Y₁T₂O₃Polyesterase was successfully expressed in (total 50mL) Erlenmeyer flasks (500mL) composed of yeast extract (10g/L), bacto tryptone (20g/L) and glucose (30g/L), buffered with phosphate buffer (100mM, pH 6.8). Cells were incubated at 28 ℃ for 24 hours until complete consumption of glucose. The cells were centrifuged at 10,000rpm for 10 minutes, and the supernatant was used directly in the reaction.

The expression level was similar to that obtained in the case of actinomadura madurata T16-1 for keratin degradation, but its thermostability was higher. Although the enzyme produced in the keratin-degrading actinomadura T16-1 lost 78% of its activity after 5 hours at 60 ℃, the enzyme expressed in yarrowia lipolytica was fully active after the same treatment.

10B-polyesterase recombinantly expressed in Bacillus subtilis SEQ ID NO 1

The polyesterase of SEQ ID NO 1 was cloned and expressed in Bacillus subtilis as described in the commercial Takara kit. The sequence of the propeptide corresponding to the sequence of the gene followed by expression of the mature polyesterase was optimized for the codon usage of Bacillus subtilis. This sequence was integrated downstream of the secretion signal sequence of B.subtilis.

The expression level was similar to that obtained in the case of the keratin degrading actinomadura T16-1.

10C-polyesterase of SEQ ID NO 1 recombinantly expressed in E.coli

The polyesterase of SEQ ID NO 1 was cloned and expressed in E.coli and more specifically in BL21, Origami and Rosetta strains. The sequence of the propeptide corresponding to the sequence of the gene followed by expression of the mature polyesterase was optimized for the codon usage of E.coli. This sequence is integrated downstream of the PelB signal sequence or the gene encoding maltose binding protein for achieving periplasmic expression, with or without a histidine tag.

The expression level was 2 to 3 times higher than that obtained in the case of actinomadura madurata T16-1 for keratin degradation.

Using a histidine tag, the protein was purified and tested for PLA depolymerization at 250- ­ 500 μm powder using PLA (Ingeo 7001D). Compared to the enzyme expressed in the keratin-degrading actinomadura T16-1, the enzyme produced in escherichia coli exhibited the same specific activity.

Example 11: biodegradable plastic compounds producing polypeptides containing SEQ ID NO 1

Method for producing 11A-plastic compound

A plastic compound was prepared comprising a solid formulation of PLA polymer (polylactic acid PLE 003 from Natureplast) and the polypeptide of SEQ ID NO:1 in pelletized form previously dried at 65 ℃ for 4 hours.

The polypeptide solid formulation was previously prepared according to the following steps: culturing a microorganism producing the polypeptide, filtering the culture, followed by ultrafiltration and diafiltration using a 3kD membrane, adding 10g.l maltodextrin, and nebulizing the mixture to obtain the polypeptide in dry powder form.

A compounder or co-rotating twin screw extruder ("Coperion ZSK 18 megalab") was used. This compounder comprises, in order, a first feeding element, two mixing elements and a second feeding element. The compounder comprises 9 successive heating zones Z1 to Z9, where the temperature can be controlled and regulated independently. Another zone Z10 exists after zone Z9, corresponding to the head of the twin screw.

According to this experiment, 96 wt% PLA was mixed with 4 wt% of a liquid formulation of the PLA depolymerase of SEQ ID NO:1 and extruded, wherein the temperature profile is described in table 3 below.

Table 3: temperature profile of compounder

Region(s)

Z1

Z2

Z3

Z4

Z5

Z6

Z7

Z8

Z9

Z10 (head)

T℃

135℃

150℃

170℃

180℃

140℃

140℃

140℃

140℃

140℃

140℃

PLA was introduced into the main hopper (before the zone Z1) at a flow rate of 9.6 kg/h. PLA passes through zones Z1-Z5, where the temperature is increased up to 180 ℃ (Z4), producing molten PLA. The enzyme was then introduced into Z6 through a side feeder N.cndot.2 at a flow rate of 0.4kg/h, where the temperature was reduced to 140 ℃.

The enzyme and PLA were then mixed together by rotation of the twin screw at 200Rpm from zone Z7 to zone Z9. The residence time from Z1 to Z9 was about 1 minute 30 seconds. The mixture of PLA and biological entities then reaches a screw head (Z10) comprising two holes of 2.5mm diameter, where the mixture is pushed to form pellets, which are then cooled and dried in water, followed by conditioning.

A plastic compound was obtained in granulated form containing 96 wt.% PLA and 4 wt.% of a formulation of PLA depolymerase of SEQ ID NO: 1. The plastic compound may be used to produce plastic articles by any technique known per se in the art.

11B-degradation test of Plastic Compounds comprising PLA and the polypeptide of SEQ ID NO: 1.

Comparative tests of different biodegradability have been carried out using the following:

a plastic compound produced according to example 11A, containing 96% by weight of PLA and 4% by weight of a formulation of a PLA depolymerase of the invention

PLA compound produced as described in example 11A, containing 100% by weight of PLA (i.e.deprived of PLA depolymerase), referred to as control

The tests were carried out at different temperatures: 28 ℃, 37 ℃ or 45 ℃.

Approximately 1 gram of PLA was placed in 100mL Tris buffer (pH 9.5). The amount of PLA was accurately measured to evaluate the theoretical amount of lactic acid produced.

Biodegradability of the compounds was evaluated by measuring the conversion of PLA, more specifically the depolymerization of PLA to lactic acid or to dimers of lactic acid. This conversion was followed by HPLC analysis.

The results (fig. 8) show that PLA compounds incorporating PLA depolymerases show greater rates of depolymerization (i.e., biodegradability) compared to control PLA compounds. Depolymerization of the PLA compound at 37 ℃ or 45 ℃ was even better compared to at 28 ℃.

Sequence listing

<110> Calbeouse Co

French national institute of agricultural food and Environment

National institute of applied sciences of France

French national research center

<120> polypeptide having polyester-degrading activity and use thereof

<130> B1898PC00

<160> 5

<170> PatentIn 3.5 edition

<210> 1

<211> 276

<212> PRT

<213> Artificial sequence

<220>

<223> PAM Polypeptides

<400> 1

Ala Thr Gln Asn Asn Pro Pro Ser Trp Gly Leu Asp Arg Ile Asp Gln

1 5 10 15

Thr Asn Leu Pro Leu Ser Arg Ser Tyr Thr Tyr Asn Ser Thr Gly Ala

20 25 30

Gly Val Asn Ala Tyr Ile Ile Asp Thr Gly Ile Tyr Thr Ala His Ser

35 40 45

Asp Phe Gly Gly Arg Ala Thr Asn Val Tyr Asp Ala Leu Gly Gly Asn

50 55 60

Gly Gln Asp Cys Asn Gly His Gly Thr His Val Ala Gly Thr Val Gly

65 70 75 80

Gly Ala Ala Tyr Gly Val Ala Lys Ala Val Asn Leu Arg Gly Val Arg

85 90 95

Val Leu Asn Cys Ser Gly Ser Gly Thr Thr Ser Gly Val Ile Ala Gly

100 105 110

Met Asn Trp Val Ala Ser Asn His Val Lys Pro Ala Val Ala Asn Met

115 120 125

Ser Leu Gly Gly Gly Tyr Ser Ser Ser Leu Asn Thr Ala Ala Asn Asn

130 135 140

Leu Ala Ser Ser Gly Val Phe Leu Ala Val Ala Ala Gly Asn Glu Thr

145 150 155 160

Thr Asn Ala Cys Asn Arg Ser Pro Ala Ser Ala Ala Asn Ala Thr Thr

165 170 175

Val Ala Ala Ser Thr Ser Thr Asp Ala Arg Ala Ser Tyr Ser Asn Tyr

180 185 190

Gly Ser Cys Val His Leu Tyr Ala Pro Gly Ser Ser Ile Thr Ser Ala

195 200 205

Trp Leu Asn Gly Gly Thr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr

210 215 220

Pro His Val Ala Gly Thr Ala Ala Leu Tyr Lys Ala Thr Tyr Gly Asp

225 230 235 240

Ala Ser Phe Ser Thr Ile Arg Ser Trp Leu Val Ser Asn Ala Thr Ser

245 250 255

Gly Val Ile Thr Gly Asn Val Ser Gly Thr Pro Asn Leu Leu Leu Asn

260 265 270

Lys Arg Ser Leu

275

<210> 2

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<223> PAM nucleic acid sequence

<400> 2

gccacgcaga acaacccgcc gtcgtggggc ctggaccgca tcgaccagac gaacctgccg 60

ctgtcgcgca gctacaccta caattccacc ggcgcgggcg tgaacgccta catcatcgac 120

accggcatct acaccgcgca ctccgacttc ggcggccgcg ccaccaacgt ctacgacgcc 180

ctcggcggca acggccagga ctgcaacggc cacggcaccc acgtcgcggg caccgtcggc 240

ggcgccgcct acggcgtggc caaggcggtc aacctgcgcg gcgtgcgcgt gctcaactgc 300

agcggcagcg gcaccacctc cggtgtcatc gccggcatga actgggtggc cagcaaccac 360

gtcaagcccg ccgtggcgaa catgtcgctg ggcggcggct actcctcctc cctgaacacg 420

gccgccaaca acctggccag ctccggcgtg ttcctggccg tcgccgcggg caacgagacc 480

accaacgcct gcaaccgctc gccggccagc gccgccaacg ccaccacggt cgccgcgagc 540

accagcaccg acgcccgggc ctcctacagc aactacggct cgtgcgtcca cctgtacgcg 600

cccggctcgt ccatcacctc cgcctggctg aacggcggca ccaacaccat cagcggcacg 660

tcgatggcca cgccgcacgt ggccgggacc gccgccctct acaaggcgac ctacggcgac 720

gcctcgttca gcaccatccg cagctggctg gtcagcaacg ccacctccgg cgtcatcacc 780

ggcaacgtgt cgggcacccc gaacctgctg ctgaacaagc gctccctg 828

<210> 3

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> N-terminal polypeptide sequence

<400> 3

Ala Thr Gln Asn Asn Pro Pro Ser Trp Gly Leu Asp Arg Ile Asp Gln

1 5 10 15

Thr Asn Leu Pro Leu Ser Arg Ser Tyr Thr Tyr Asn

20 25

<210> 4

<211> 1161

<212> DNA

<213> Artificial sequence

<220>

<223> DNA sequence

<400> 4

atgagacgac gtaccctgcc catcgccgtc ctcgccgccg ttcccctggc cgtggcgggc 60

gccctgcccg ccggagccgc ccccgccgcc cccgccgtcc cggtcgccat ggcggccgcc 120

ggacagggcg tcgccggaca gtacatcgtg acgctgaaga agggcgtctc ggtcgactcg 180

accgtcgcca agcgcggaat ccgcacccag caccgtttcg gcaaggtgct gaacggcttc 240

tccgccaagc tcaccgatga ccaactgtcc aagctgcgca ccacgcccgg tgtcgcgtcc 300

atcgagcagg acgccgtcat cacggtggac gccacgcaga acaacccgcc gtcgtggggc 360

ctggaccgca tcgaccagac gaacctgccg ctgtcgcgca gctacaccta caattccacc 420

ggcgcgggcg tgaacgccta catcatcgac accggcatct acaccgcgca ctccgacttc 480

ggcggccgcg ccaccaacgt ctacgacgcc ctcggcggca acggccagga ctgcaacggc 540

cacggcaccc acgtcgcggg caccgtcggc ggcgccgcct acggcgtggc caaggcggtc 600

aacctgcgcg gcgtgcgcgt gctcaactgc agcggcagcg gcaccacctc cggtgtcatc 660

gccggcatga actgggtggc cagcaaccac gtcaagcccg ccgtggcgaa catgtcgctg 720

ggcggcggct actcctcctc cctgaacacg gccgccaaca acctggccag ctccggcgtg 780

ttcctggccg tcgccgcggg caacgagacc accaacgcct gcaaccgctc gccggccagc 840

gccgccaacg ccaccacggt cgccgcgagc accagcaccg acgcccgggc ctcctacagc 900

aactacggct cgtgcgtcca cctgtacgcg cccggctcgt ccatcacctc cgcctggctg 960

aacggcggca ccaacaccat cagcggcacg tcgatggcca cgccgcacgt ggccgggacc 1020

gccgccctct acaaggcgac ctacggcgac gcctcgttca gcaccatccg cagctggctg 1080

gtcagcaacg ccacctccgg cgtcatcacc ggcaacgtgt cgggcacccc gaacctgctg 1140

ctgaacaagc gctccctgta a 1161

<210> 5

<211> 386

<212> PRT

<213> Artificial sequence

<220>

<223> polypeptide sequence

<400> 5

Met Arg Arg Arg Thr Leu Pro Ile Ala Val Leu Ala Ala Val Pro Leu

1 5 10 15

Ala Val Ala Gly Ala Leu Pro Ala Gly Ala Ala Pro Ala Ala Pro Ala

20 25 30

Val Pro Val Ala Met Ala Ala Ala Gly Gln Gly Val Ala Gly Gln Tyr

35 40 45

Ile Val Thr Leu Lys Lys Gly Val Ser Val Asp Ser Thr Val Ala Lys

50 55 60

Arg Gly Ile Arg Thr Gln His Arg Phe Gly Lys Val Leu Asn Gly Phe

65 70 75 80

Ser Ala Lys Leu Thr Asp Asp Gln Leu Ser Lys Leu Arg Thr Thr Pro

85 90 95

Gly Val Ala Ser Ile Glu Gln Asp Ala Val Ile Thr Val Asp Ala Thr

100 105 110

Gln Asn Asn Pro Pro Ser Trp Gly Leu Asp Arg Ile Asp Gln Thr Asn

115 120 125

Leu Pro Leu Ser Arg Ser Tyr Thr Tyr Asn Ser Thr Gly Ala Gly Val

130 135 140

Asn Ala Tyr Ile Ile Asp Thr Gly Ile Tyr Thr Ala His Ser Asp Phe

145 150 155 160

Gly Gly Arg Ala Thr Asn Val Tyr Asp Ala Leu Gly Gly Asn Gly Gln

165 170 175

Asp Cys Asn Gly His Gly Thr His Val Ala Gly Thr Val Gly Gly Ala

180 185 190

Ala Tyr Gly Val Ala Lys Ala Val Asn Leu Arg Gly Val Arg Val Leu

195 200 205

Asn Cys Ser Gly Ser Gly Thr Thr Ser Gly Val Ile Ala Gly Met Asn

210 215 220

Trp Val Ala Ser Asn His Val Lys Pro Ala Val Ala Asn Met Ser Leu

225 230 235 240

Gly Gly Gly Tyr Ser Ser Ser Leu Asn Thr Ala Ala Asn Asn Leu Ala

245 250 255

Ser Ser Gly Val Phe Leu Ala Val Ala Ala Gly Asn Glu Thr Thr Asn

260 265 270

Ala Cys Asn Arg Ser Pro Ala Ser Ala Ala Asn Ala Thr Thr Val Ala

275 280 285

Ala Ser Thr Ser Thr Asp Ala Arg Ala Ser Tyr Ser Asn Tyr Gly Ser

290 295 300

Cys Val His Leu Tyr Ala Pro Gly Ser Ser Ile Thr Ser Ala Trp Leu

305 310 315 320

Asn Gly Gly Thr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His

325 330 335

Val Ala Gly Thr Ala Ala Leu Tyr Lys Ala Thr Tyr Gly Asp Ala Ser

340 345 350

Phe Ser Thr Ile Arg Ser Trp Leu Val Ser Asn Ala Thr Ser Gly Val

355 360 365

Ile Thr Gly Asn Val Ser Gly Thr Pro Asn Leu Leu Leu Asn Lys Arg

370 375 380

Ser Leu

385

Claims (15)

1. Use of a polypeptide for degrading a polyester-containing material, wherein the polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90% or 92% identity to the full-length amino acid sequence set forth in SEQ ID No. 1, the polypeptide comprising a biologically active portion comprising a catalytic domain comprising the amino acids His 71, Asp 40 and Ser 221, a calcium binding site comprising the amino acids Ala 172, Ala 174, His 197 and/or the amino acids Asp 12, Asp 15, Gln 16, and a disulfide bond comprising the amino acids Cys 68, Cys 100, Cys164 and Cys 195, and having polyester degrading activity.

2. Use according to claim 1, wherein the polyester containing material is a polylactic acid (PLA) containing material.

3. Use according to claim 2, wherein the polyester containing material is a PLLA containing material.

4. The use of claim 1 or 2, wherein the polyester-containing material is contacted with the polypeptide to depolymerize to monomers, oligomers, or mixtures thereof, at least one polyester of the polyester-containing material.

5. A method of producing monomers, oligomers or mixtures thereof from a polyester containing material, said method comprising

-exposing the polyester containing material to a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90% or 92% identity to the full length amino acid sequence as set forth in SEQ ID NO 1, the polypeptide comprising a biologically active portion comprising a catalytic domain comprising the amino acids His 71, Asp 40 and Ser 221, a calcium binding site comprising the amino acids Ala 172, Ala 174, His 197 and/or the amino acids Asp 12, Asp 15, Gln 16 and a disulfide bond comprising the amino acids Cys 68, Cys 100, Cys164 and Cys 195 and having polyester degrading activity,

-recovering the monomer, oligomer or mixture thereof.

6. Use of a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90% or 92% identity to the full-length amino acid sequence set forth in SEQ ID No. 1, for the preparation of a polyester-containing material, the polypeptide comprising a biologically active portion comprising a catalytic domain comprising the amino acids His 71, Asp 40 and Ser 221, a calcium binding site comprising the amino acids Ala 172, Ala 174, His 197 and/or the amino acids Asp 12, Asp 15, Gln 16, and a disulfide bond comprising the amino acids Cys 68, Cys 100, Cys164 and Cys 195, and having polyester degrading activity.

7. Use according to claim 6, wherein the polyester containing material is a plastic compound.

8. A polyester-containing material comprising at least one polyester and a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90% or 92% identity to the full-length amino acid sequence set forth in SEQ ID No. 1, the polypeptide comprising a biologically active portion comprising a catalytic domain comprising amino acids His 71, Asp 40 and Ser 221, a calcium binding site comprising amino acids Ala 172, Ala 174, His 197 and/or amino acids Asp 12, Asp 15, Gln 16, and a disulfide bond comprising amino acids Cys 68, Cys 100, Cys164 and Cys 195, and having polyester degrading activity.

9. The polyester-containing material of claim 8, wherein the polyester is PLA.

10. The polyester-containing material of claim 9, wherein the polyester is PLLA.

11. The polyester-containing material of claim 8 or 9, which is a plastic compound.

12. A plastic article comprising the polyester-containing material of claim 11.

13. A method for preparing the polyester-containing material of claim 8, comprising the step of mixing a polyester with a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90% or 92% identity to the full-length amino acid sequence set forth in SEQ ID No. 1, the polypeptide comprising a biologically active portion comprising a catalytic domain comprising the amino acids His 71, Asp 40 and Ser 221, a calcium binding site comprising the amino acids Ala 172, Ala 174, His 197 and/or the amino acids Asp 12, Asp 15, Gln 16, and a disulfide bond comprising the amino acids Cys 68, Cys 100, Cys164 and Cys 195, and having polyester degrading activity, wherein the step of mixing is performed at a temperature at which the polyester is in a partially or fully molten state.

14. The method of claim 13, wherein the polypeptide is mixed with a polyester at a temperature between the glass transition temperature and the melting point of the polyester or at a temperature corresponding to the melting point of the polyester or higher.

15. The method of claim 13, wherein the polypeptide and polyester are mixed during an extrusion process.

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